Thrombin binds to its substrate fibrinogen in the central amino-terminal region and cleaves fibrinopeptides A and B from the A.alpha. and B.beta. chains, respectively, converting fibrinogen to fibrin. The thrombin-fibrinogen binding interaction is mediated through an anion-binding fibrinogen recognition exosite in thrombin (Fenton, J. W., II, et al., Biochemistry 27:7106-7112, 1988; Binnie, C. G. and Lord, S. T., Blood 81:3186-3192, 1993; Stubbs, M. T. and Bode, W., Thromb. Res. 69:1-58, 1993) that is situated in an extended patch of positively charged residues in the region of the thrombin loop segment centered around Lys 70-Glu 80 (Noe, G., et al., J. Biol. Chem. 263:11729-11735, 1988). The exosite also binds to heparin cofactor II (Church, F. C., et al., J. Biol. Chem. 264:18419-18425, 1989), factor V and factor VII (Esmon, C. T. and Lollar P., J. Biol. Chem. 271:13882-13887, 1996) the platelet or endothelial cell thrombin receptor (Herbert, J.-M., et al., Biochem. J. 303:227-231, 1994), thrombomodulin (Tsiang, M., et al., Biochemistry 29:10602-10612, 1990; Suzuki, K. and Nishioka, J., J. Biol. Chem. 266:18498-18501, 1991), GPIb.alpha. (De Marco, L., et al., J. Biol. Chem. 269:6478-6484, 1994), as well as to a strongly anionic sequence in the carboxyl-terminal region of the leech thrombin-inhibitor, hirudin (Dodt, J., et al., FEBS Letters 165:180-184, 1984; Maraganore, J. M., et al., J. Biol. Chem. 264:8692-8698, 1989; Bourdon, P., et al., Biochemistry 29:6379-6384, 1990; Rydel, T. J., et al., Science 249:277-280, 1990; Naski, M., et al., J. Biol. Chem. 265:13484-13489, 1990; Parry, M. A. A., et al., Biochemistry 33:14807-14814, 1994).
In addition to binding to fibrinogen at its substrate site, thrombin binds to fibrin at a `non-substrate` site(s) (Fenton, J. W., II, et al., supra, 1988; Binnie, C. G. and Lord, S. T., supra, 1993; Berliner, L. J., et al., Biochemistry 24:7005-7009, 1985; Kaczmarek, E. and McDonagh, J., J. Biol. Chem. 263:13896-13900, 1988; Vali, Z. and Scheraga, H. A., et al., Biochemistry 27:1956-1963, 1988). It is commonly believed that non-substrate binding takes place at the same location as fibrinogen substrate binding, namely the central E domain. As determined from binding experiments with .sup.125 I-thrombin by Liu, et al. (Liu, C. Y., et al., J. Biol. Chem. 254:10421-10425, 1979) two classes of non-substrate sites exist in fibrin, one of `high` affinity (K.sub.a, .about.6.times.10.sup.5 M.sup.-1) and the other of `low` affinity (K.sub.a, .about.7.times.10.sup.4 M.sup.-1). Hogg and Jackson also found two classes of sites in fibrin with affinity constants of 3.3.times.10.sup.6 and 3.0.times.10.sup.4, respectively (Hogg, P. J. and Jackson, C. M., J. Biol. Chem. 265:241-247, 1990). It has been inferred from available information that all non-substrate thrombin binding, especially that of high affinity, is in the E domain (Binnie, C. G. and Lord, S. T., supra, 1993), although to our knowledge this subject has not been specifically addressed.
Human fibrinogen is chromatographically separable into two major components (`peak 1` and `peak 2`) which differ with respect to the structure of their .gamma. chains (Mosesson, M. W., et al., J. Biol. Chem. 247:5223-5227, 1972). Dimeric peak 1 fibrinogen molecules each contain two .gamma..sub.A chains (.gamma.1-411V) whereas peak 2 fibrinogen molecules, which amount to .about.15% of the total fibrinogen population (Mosesson, M. W. and Finlayson, J. S., J. Lab. Clin. Med. 62:663-674, 1963), have one .gamma..sub.A and one .gamma.' chain (.gamma.1-427L) (Wolfenstein-Todel, C. and Mosesson, M. W., Proc. Natl. Acad. Sci. USA 77:5069-5073, 1980; Mosesson, M. W., Ann. NY Acad. Sci. 408:97-113, 1983). Similar .gamma. chain variants have been identified in rodent (Crabtree, G. R. and Kant, J. A., Cell 31:159-166, 1982; Legrele, C. D., et al., Biochem. Biophys. Res. Commun. 105:521-529, 1982) and bovine (Agnes Henschen, personal communication) fibrinogens and may exist in other animal species as well (Finlayson, J. S. and Mosesson, M. W., Biochim. Biophys. Acta 82:415-417, 1964). In humans, .gamma.' chains arise through alternative processing of the primary mRNA transcript (Chung, D. W. and Davie, E. W., Biochemistry 23:4232-4236, 1984), and differ structurally in their C-terminal sequences in that .gamma..sub.A chain residues 408 to 411 are replaced in .gamma.' chains by an anionic 20 amino acid sequence (Mosesson, M. W., supra, 1983; Wolfenstein-Todel, C. and Mosesson, M. W., Biochemistry 20:6146-6149, 1981). In rats (Crabtree, G. R. and Kant, J. A., supra, 1982; Homandberg, G. A., et al., Thromb. Res. 39:263-269, 1985) and cows (Agnes Henschen, personal communication) .gamma..sub.A 408 to 411 is replaced by a shorter but homologous sequence (see Table 1 below). The rat and human .gamma.' chains are tyrosine-sulfated at .gamma.' 418 (Hortin, G. L., Biochemistry International 19:1355-1362, 1989; Hirose, S., et al., J. Biol. Chem. 263:7426-7430, 1988) and also at .gamma.' 422 in humans (Agnes Henschen, personal communication).
TABLE 1 ______________________________________ Carboxyl-terminal sequences of .gamma. chains and hirudin. Homologous position are outfitted Chain (position) Amino Acid Sequence ______________________________________ 1 #STR1## ______________________________________
.gamma..sub.A and .gamma.' chains are functionally equivalent with respect to factor XIIIa-catalyzed crosslinking (Wolfenstein-Todel, C. and Mosesson, M. W., supra, 1980), but unlike the .gamma..sub.A chain, .gamma.' chains lack the complete platelet binding sequence, .gamma.' .sub.A 400-411, and therefore do not support ADP-induced fibrinogen binding or platelet aggregation (Harfenist, E. J., et al., Blood 64:1163-1168, 1984; Kirschbaum, N. E., et al., Blood 79:2643-2648, 1992; Farrell, D. H. and Thiagarajan, P., J. Biol. Chem. 269:226-231, 1994). Siebenlist, et al. has recently presented evidence that plasma factor XIII binds specifically to .gamma.' chains (Siebenlist, K. R., et al., Biochemistry, 35:10448-10453, 1996), but little else is known about its functions.
In other studies, workers have investigated the relationship between hirudin and thrombin inhibition. Hirudin is a naturally occurring polypeptide produced in the salivary glands of the blood sucking leech Hirudo medicinalis. Hirudin is a potent anticoagulant which binds tightly to thrombin in a two-step process. Initially hirudin binds, via its carboxyl-terminal 53-65 amino acid residues (hirugen), to the anion-binding exosite of thrombin. Subsequently, the amino-terminal region of hirudin binds to the catalytic site of thrombin. Numerous artificial constructs have been devised to mimic the inhibitory action of hirudin, such as hirulog and hirutonin. These hirudin-analogues are comprised of an amino-terminal segment, which binds the catalytic site of thrombin, linked to the hirugen sequence. The various analogues (hirulog 1, hirutonin 2, etc.) result from substitutions in each of these segments in different combinations.
In the Examples below we present compelling evidence that the anionic carboxy-terminal .gamma.' chain sequence situated in the fibrin D domain constitutes the high-affinity thrombin binding site, which is itself separate and distinct from the low affinity thrombin binding sites that reside in the central E domain. This finding has relevance in terms of designing an improved thrombin inhibitor.